2016 Volume 64 Issue 8 Pages 1092-1098
The transfer of urea from a urea formulation to the stratum corneum varies with the formulation base and form, and impacts the formulation’s therapeutic effect. Consequently, determining the amount of urea transferred is essential for developing efficient formulations. This study assessed a simple method for measuring the amount of urea accumulated in the stratum corneum. Conventional methods rely on labeling urea used in the formulation with radiocarbon (14C) or other radioactive isotopes (RIs), retrieving the transferred urea from the stratum corneum by tape stripping, then quantitating the urea. The handling and use of RIs, however, is subject to legal regulation and can only be performed in sanctioned facilities, so methods employing RIs are neither simple nor convenient. We therefore developed a non-radiolabel method “tape stripping-colorimetry (T-C)” that combines tape stripping with colorimetry (urease–glutamate dehydrogenase (GLDH)) for the quantitative measurement of urea. Urea in the stratum corneum is collected by tape stripping and measured using urease-GLDH, which is commonly used to measure urea nitrogen in blood tests. The results indicate that accurate urea measurement by the T-C method requires the application of 1400 mg (on hairless rats) of a 20% urea solution on a 50 cm2 (5×10 cm) area. Further, we determined the amount of urea accumulated in the stratum corneum using formulations with different urea concentrations, and the time course of urea accumulation from formulations differing in the rate of urea crystallization. We demonstrate that the T-C method is simple and convenient, with no need for 14C or other RIs.
Urea improves the water-retention capability of the skin1–4) and is widely used as a therapeutic agent in formulations for keratoderma, senile xerosis, and other dry-skin diseases.5) Urea is percutaneously absorbed following dermal application6) and has a keratin-softening and moisture-retention effect on the stratum corneum when taken up and retained there. The keratin-softening effect increases with increasing urea content in externally applied formulations and with increasing urea uptake by the stratum corneum.7) The effects of the formulation base and form on the course of percutaneous absorption and transfer to the stratum corneum are readily apparent. It is accordingly essential to determine the amount of urea transferred to the stratum corneum during urea formulation development. We here assessed a simple and convenient new method for measuring urea transfer.
Many studies have described the use of tape stripping to retrieve urea from the stratum corneum,8–14) and radiocarbon (14C)-labeled urea is widely used for determining the amount of retrieved urea. Radioisotopes (RIs) are widely used and highly effective for analyses in the life sciences and many other fields of research,15,16) but they also involve the risk of mishandling, leading to injury of the researcher and other persons in the vicinity, and to environmental dispersion and contamination. The use of RIs in Japan is therefore strictly regulated; experiments requiring RIs can only be performed in legally approved facilities and are subject to numerous regulations. Methods requiring RI labeling therefore cannot be regarded as simple or convenient.
We embarked on developing a simple and convenient non-radiolabel method to quantify urea accumulated in the stratum corneum. In this method (the “tape stripping-colorimetry (T-C) method”), the urea transferred to and retained in the stratum corneum is retrieved by tape stripping and is then measured by colorimetry (urease–glutamate dehydrogenase (GLDH)),17–22) a measurement technique commonly used in blood tests. We here investigated the amount of formulation that must be applied for accurate measurement of the accumulated urea using the T-C method, used these conditions to show the relationship between the concentration of urea in the formulation (10 or 20%) and the quantity of urea accumulated in the stratum corneum, and assessed the effectiveness of the T-C method as a means of evaluating formulations.
In the first study in this series (Deposits from Creams Containing 20% (w/w) Urea and Suppression of Crystallization (Part 1)), we found that in a simulated low-humidity winter environment (40–50% relative humidity (RH), 20–25°C), urea crystal formation occurs more slowly in lecithin (LEC) formulations than in non-ionic surfactant (NS) formulations. The rate of urea crystallization in formulations affects the rate of urea transfer to the stratum corneum.23–25) Therefore, in the present study, we used the T-C method to investigate the time course of urea accumulation from LEC and NS formulations known to exhibit different rates of urea crystallization. The results show that the T-C method provides a simple, convenient, and effective means of measuring accumulated urea without the use of radiocarbon.
The application samples were selected from typical O/W (oil in water) creams to provide substantially different rates of urea crystallization based on our first report in this series. The model formulations were: (1) a rapid-crystallization 20% urea formulation containing NS as the emulsifier (20% NS; Company A); (2) a slow-crystallization 20% urea developmental formulation containing LEC as the emulsifier (20% LEC; Company B); and (3) a 10% urea developmental formulation (10% LEC; Company B) based on the 20% LEC formulation but with a reduced urea content.
T-C MethodThe formulation sample is evenly applied to a specified area of skin on hairless rats and dried for 10–30 min with cold-air drying to simulate a dry winter-season environment. The stratum corneum is then subjected to 10 successive tape strippings at given intervals, and the urea content on each strip and the total urea content for all 10 strips are determined by colorimetry (urease–GLDH) (Fig. 1).
The amount of sample applied was specified by the product of the unit-area quantity of approximately 28 mg/cm2 and the application area. This was based on a report by Fukahori et al.25) in which 50 mg of 14C-urea cream was applied to 1.77 cm2, thus providing a unit-area quantity of 28.25 mg/cm2.
(1) Sample Application Conditions and Tape StrippingAt given intervals following sample application, the surface sample residue was removed by wiping the designated skin area four times with absorbent cotton moistened with distilled water (Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan) heated to 40°C. The specimen area was dried using an air-blow drier, then a patch of vinyl tape (Nitto Denko Corp., Osaka, Japan) the same size as the specimen area was applied to the area and removed to obtain the specimen strip. The tape-stripping process was repeated 10 times to provide 10 successive stratum corneum strips. Two pieces of tape of the same size (not used for tape-stripping) were used for blank measurements.
(2) Determination of the Amount of Urea on the Stratum Corneum StripsEach tape sample was immersed in 15 mL of ethanol in a sample bottle, sealed, and left to stand for 1 d. The container contents were agitated for approximately 30 min on a strong shaker (ReciprocalShaker SR-II; Titech Corp., Saitama, Japan), centrifuged and then separated into 10 mL fractions in glass test tubes from 15 mL. The ethanol was removed using nitrogen gas, 1 mL of distilled water was added, and the tube was vortexed to ensure that the solution contacted the entire inner surface of the tube. The tube was then centrifuged (3000 r/min, 15 min), and the amount of urea in the liquid fraction was measured.
The amount of urea was measured by urease–GLDH absorbance (measurement wavelength: 340 nm) on a biochemical autoanalyzer (AU400; Olympus Corp., Tokyo, Japan). The absorbance reading was used to calculate the amount of urea per application area.
Determination of the Rate of Urea Crystallization in the Model FormulationsThe rate of urea crystallization in the model formulations (20% NS, 20% LEC) was determined by microscopic observation of urea crystals from each formulation using a simple glass-surface application method.2)
Approximately 20 mg of each formulation sample was spread evenly using an ointment spatula to obtain a uniform thin film covering the entire surface of a glass slide (60×26 mm in area), left to stand at 40–55% RH (20–25°C) for 0 (at the start), 15, 60, or 120 min, then was observed under a polarizing microscope (BH, BX-10; Olympus Corp., Tokyo, Japan; ×100) to allow accurate visualization of the urea crystals.
Quantitative Determination of Urea Accumulation in the Stratum Corneum by the T-C MethodApplication Area, Quantity, and Measurement Methods of Accumulated UreaThe amount of formulation that must be applied to hairless rats in order to quantify the urea accumulated in the stratum corneum using the T-C method was assessed based on the report by Fukahori et al.25): each formulation sample was spread evenly over the application area to obtain a uniform thin film covering the entire skin surface on 50 cm2 area (1400 mg). We choosed the largest application area (50 cm2: 5×10 cm) that can be get without being influenced by the difference of sizes of hairless rats (Table 1). The amount of accumulated urea was determined using the T-C method by colorimetric quantification of the 10 tape strips applied to the skin and then stripped off 5 h after applying the cream.
| Model formulation | Application area | Quantity | Number (animal No.) |
|---|---|---|---|
| 20% LEC formulation | 50 cm2×1 site | 1400 mg×1 site | 2 (101–102) |
| No application | 50 cm2×1 site | — | 1 (401) |
In the measurement by colorimetry, the reagent (Ltype wakoUN2, Wako Pure Chemical Industries, Ltd., Osaka, Japan) was used for the urease–GLDH. Measurement range of urea by the reagent is in the 0.81–428 mg /dL. In terms of “μg/cm2,” the amount is a 0.24–128 μg/cm2. The amount of urea at the stratum corneum was obtained by the subtraction of urea of the tape from the measured value.
The tape that was used to tape stripping is vinyl tape (Nitto Denko Corp., Osaka, Japan). The formulation used in experiments is 20% LEC formulation. The animal test system is “3 male HWY/Slc hairless rats (age 9 weeks).”
Amount of Urea Accumulation Using Formulations with Different Urea ConcentrationsThe amount of urea accumulated in the stratum corneum using formulations with different urea concentrations was measured using 10% LEC and 20% LEC applied to hairless rats. In both cases, 1400 mg of formulation was applied to 50 cm2 (5×10 cm) and dried with an air-blow drier for 30 min, simulating a dry winter-season environment (Table 2). The accumulated urea was measured by the T-C method 5 h after drying, with 10 tape strippings of the application site followed by colorimetric determination of the urea on each strip and the total urea on the 10 strips. In the measurement by colorimetry, the reagent (Ltype wakoUN2, Wako Pure Chemical Industries, Ltd., Osaka, Japan) was used for the urease–GLDH. The tape that was used to tape stripping is vinyl tape (Nitto Denko Corp., Osaka, Japan). The formulations used in experiments are 10% LEC formulation and 20% LEC formulation. The animal test system is “12 male HWY/Slc hairless rats (age 9 weeks), 2 groups, 6 rats per group.”
| Group | Model formulation | Application area | Quantity | Number (animal No.) |
|---|---|---|---|---|
| 1 | No application | 50 cm2×1 site | — | 6 (101–106) |
| 20% LEC formulation | 50 cm2×1 site | 1400 mg×1 site | ||
| 2 | No application | 50 cm2×1 site | — | 6 (201–206) |
| 10% LEC formulation | 50 cm2×1 site | 1400 mg×1 site |
The time course of urea accumulation in the stratum corneum was measured using the T-C method following application of two formulations (20% NS and 20% LEC) with different bases and rates of urea crystal formation. In both cases, 1400 mg of the formulation was applied to 50 cm2 (5×10 cm) application site on each hairless rat (Table 3). The applied formulation was dried with an air-blow drier for 10 min. The accumulated urea was measured using the T-C method at 2.5, 5, and 10 h after drying each with 10 strippings of the application site followed by colorimetric determination of the amount of urea on each strip and the total amount on all 10 strips. In the measurement by colorimetry, the reagent (Ltype wakoUN2, Wako Pure Chemical Industries, Ltd.) was used for the urease–GLDH. The tape that was used to tape stripping is vinyl tape (Nitto Denko Corp.). The formulations used in experiments are 20% NS and 20% LEC. The animal test system is “36 male HWY/Slc hairless rats (age 9 weeks), 6 groups, 6 rats per group.”
| Group | Model formulation | Application area | Quantity | Time (h) | Number (animal No.) |
|---|---|---|---|---|---|
| 1 | No application | 50 cm2×1 site | — | 2.5 | 6 (101–106) |
| LEC formulation | 50 cm2×1 site | 1400 mg×1 site | |||
| 2 | No application | 50 cm2×1 site | — | 5 | 6 (201–206) |
| LEC formulation | 50 cm2×1 site | 1400 mg×1 site | |||
| 3 | No application | 50 cm2×1 site | — | 10 | 6 (301–306) |
| LEC formulation | 50 cm2×1 site | 1400 mg×1 site | |||
| 4 | No application | 50 cm2×1 site | — | 2.5 | 6 (401–406) |
| NS formulation | 50 cm2×1 site | 1400 mg×1 site | |||
| 5 | No application | 50 cm2×1 site | — | 5 | 6 (501–506) |
| NS formulation | 50 cm2×1 site | 1400 mg×1 site | |||
| 6 | No application | 50 cm2×1 site | — | 10 | 6 (601–606) |
| NS formulation | 50 cm2×1 site | 1400 mg×1 site |
The urea content (µg/cm2) of each of the 10 strips and the 10-strip total for each animal was determined, the mean and standard deviation for each group was calculated, and the urea content of each of the 10 strips and the 10-strip total was compared between groups. Student’s t-test was applied and p<0.05 was considered significant.
If the urea content of a strip from an application site was less than that of the corresponding strip obtained from an unapplied site, then the urea content was taken to be zero.
Guidelines for Animal Welfare FollowedTesting Facility: Nihon Bioresearch Inc. (Gifu, Japan)Basic Guidelines for the Use of Experimental Animals in Institutions under the Jurisdiction of the Ministry of Health, Labour and Welfare (Notification No. 0601001 of the Science Bureau, Japanese Ministry of Health, Labour and Welfare, June 1, 2006)
Guidelines for Management and Welfare of Experimental Animals (Nihon Bioresearch Inc., April 2, 2007; modified on August 27, 2010)The present study was evaluated by the Committee for the Ethical Treatment of Animals and approved by Management at the test facility.
The microscopic images of formulations applied to glass slides in Fig. 2 show clear differences between the two formulations. The 20% NS formulation showed many acicular crystals 15 min after application whereas the 20% LEC formulation showed relatively few crystals even at 120 min, thus confirming that the rate of crystallization in the 20% LEC formulation is slower than in the 20% NS formulation.
At least 90% of the 1.40g of LEC formulation applied to each of two animals on a 50 cm2 area was retained as a coating (1.26 g on animal 101 and 1.30 g on animal 102). Measurements were conducted 5 h after application of the formulations to determine the cumulative amount of urea on the 10 strips applied to each animal. The results are shown in Fig. 3 and indicate that the amount of urea on successive strips decreased with increasing strip number for animal 101 but not for animal 102. A control animal (no formulation applied) was used to determine the amount of biologically-derived urea and this amount was shown to remain essentially constant.
The cumulative and total amount of urea on strips 1 to 10 after sampling the 50 cm2 test area was clearly higher for animals 101 and 102 than for the control animal, indicating that a 50 cm2 area is sufficient for assessing urea accumulation following application of a urea formulation. Unused vinyl tape contained 0.09 µg urea/cm2.
The above results clearly indicate that an application area of 50 cm2 (5×10 cm) with an application amount of 1400 mg of a 20% (w/w) urea formulation (approximately 28 mg/cm2) is appropriate for measuring the amount of urea accumulated in the stratum corneum of hairless rats.
Urea Accumulation Following Application of 10 or 20% Urea FormulationsFigures 4–6 show the amount of urea accumulated (µg urea/cm2) in the stratum corneum following application of LEC formulations containing 10 or 20% urea. Data are presented for each successive strip, the cumulative amount for each stripping series, and the 10-strip total for each sampled site. The amount of urea tended to decrease with increasing strip number for both LEC formulations, despite fluctuations in individual animals. The mean value of the six animals in each group was higher in the 20% LEC group than in the 10% LEC group for all strips except strip 8, and there was a statistically significant difference between the group means for strips 4 to 6. The mean of the total amount of urea in strips 1–10 was significantly higher for the 20% LEC group (15.05 µg/cm2) than for the 10% LEC group (11.95 µg/cm2), as shown in Fig. 7. Unused vinyl tape contained 0.12 µg urea/cm2.
☆: p<0.05 (Student’s t-test).
☆: p<0.05 (Student’s t-test).
☆: p<0.05 (Student’s t-test).
Figures 7–10 show the amount of urea accumulated on each of the 10 strips applied to the stratum corneum following application of the 20% LEC and 20% NS formulations, and the total for all 10 strips, at 2.5, 5, and 10 h post-application.
The amount of urea per strip tended to decrease with increasing strip number at 2.5, 5, and 10 h after application, although variations were observed among the animals (Figs. 7–9). Strips 1 to 5 showed higher urea levels 2.5 h after application of the LEC formulation compared to the NS formulation, but the levels on strips 6 to 10 were higher following application of the NS formulation, but none of these differences were statistically significant. Urea levels were higher 5 h after applying the LEC formulation compared to the NS formulation except for strips 1, 4, and 10, and the difference was statistically significant for strips 3 and 5. At 10 h post-application, the NS formulation provided higher levels than the LEC formulation except for strips 5, 9, and 10, but these differences were not statistically different.
☆: p<0.05 (Student’s t-test).
None of the 10 successive strips applied to the controls showed significant differences in urea content.
The total amount of urea per unit area (µg/cm2) on the 10 strips following application of the LEC formulation to the stratum corneum was 4.83 µg/cm2 at 2.5 h after application, 9.01 µg/cm2 at 5 h, and 6.21 µg/cm2 at 10 h. Therefore, the accumulated amount of urea peaked around 5 h after application and urea accumulation decreased in the order 5<10<2.5 h. A similar time course was observed for the NS formulation: the total amount of accumulated urea was 3.74 µg/cm2 at 2.5 h, 7.43 µg/cm2 at 5 h, and 6.95 µg/cm2 at 10 h, suggesting that the amount of accumulated urea from the LEC formulation was higher than from the NS formulation at 2.5 h and 5 h after application, although the differences were not statistically significant. The NS formulation provided a slightly higher level of accumulated urea 10 h after application, but the difference was not statistically significant (Fig. 10).
The results shown using the T-C method, clearly indicate that urea transferred sooner from the slowly crystallizing LEC formulation into the skin than from the rapidly crystallizing NS formulation.
This investigation showed that urea accumulated in the stratum corneum faster from the slowly crystallizing LEC formulation than from the rapidly crystallizing NS formulation and peaked in the ten sampled upper stratum corneum layers approximately 5 h after application.
This study confirmed the utility of a new approach for determining the amount of urea accumulated in the stratum corneum. The T-C method, which combines simple tape stripping and colorimetry (urease–GLDH), is effective and does not require radiocarbon (14C) or other regulated radioisotopes.
Urea in a LEC formulation containing 20% urea applied to a 50 cm2 (5×10 cm) area (1400 mg) can be quantified by the T-C method following application on hairless rats. The application of 1400 mg of 10 or 20% urea formulations (“10% LEC” and “20% LEC”) on a 50 cm2 area, followed by quantification of the urea accumulated in the stratum corneum using the T-C method, showed that more urea was transferred from the higher concentration formulation. Specifically, following 10 tape-strippings, strips 1 to 7 and strips 9 and 10 showed a higher urea content following application of 20% LEC compared to 10% LEC. The difference between the two formulations was statistically significant in strips 4–6, and in the total for all 10 strips, consistent with the general tendency for the rate of transfer of a dissolved drug to the stratum corneum to be proportional to the concentration and the skin/cream base distribution coefficient as the skin/base partition coefficient increases.26)
The two formulations have the same base so the observed differences in the amount of urea transferred to the stratum corneum is due to their different urea concentrations,25,26) in accordance with Fick’s law of distribution.27) Thus, the results confirm the effectiveness of the T-C method.
No significant difference was observed in the time course experiment determining the rate of urea transfer to the stratum corneum from 20% LEC and 20% NS, despite their differences in the rate of urea crystallization. The LEC formulation showed higher urea transfer than the NS formulation up to 5 h after application but the NS formulation transferred more rapidly at 10 h after application. The urea accumulation levels at various times after application indicate that the LEC formulation provided faster urea transfer and, at 10 h post-application, deeper stratum corneum penetration compared to the 20% NS formulation.
These results show that urea accumulated more slowly in the stratum corneum following the application of a rapidly crystallizing formulation, such as the 20% NS, compared to formulations that tend to inhibit crystallization. Consequently, the transfer of urea as a medication into the stratum corneum and its therapeutic effect can be enhanced by maintaining the solubility of urea in the base and by minimizing its intra-formulation crystallization following application.23,24)
The results of the present study further indicate that the T-C method is safer and simpler than 14C-urea labeling, is less subject to legal regulation, is a reliable method for measuring urea transfer to the stratum corneum, and has utility for the selection of appropriate urea-containing formulations.
Urea formulations are frequently used clinically to treat keratoderma, senile xerosis, and other dry-skin disorders, and identifying formulations for the efficient transfer of urea from the base to the stratum corneum is key for high efficacy. A simple method for measuring the amount of urea transferred is highly desirable during the formulation process. We will continue research urea formulation products to refine and increase the accuracy of the T-C method, and anticipate its growing use to evaluate diverse formulations.
We are grateful to Mr. Masayoshi Shimizu at Nihon Bioresearch Inc. for providing technical support.
The authors declare no conflict of interest.
